1. Field of the Invention
The present invention relates to the method of obtaining the chromosomes more easily and efficiently than the conventional method using colhitin or its derivatives (colcemid), in more particular to the method of inducing premature chromosome condensation in interphase cell with inhibitors of serine/threonine protein phosphatases, that permit one to obtain not only mitotic chromosomes but also prematurely condensed chromosomes of interphase nuclei.
2. Description of the Related Art
Chromosomal analysis is being widely used for screening or diagnosis of genetic diseases, assay of the mutagenecity of physical or chemical factors or other various cytogenetical purposes in medical, biological, agricultural or other fields. Chromosomes are obtained usually from mitotic cells using colhitin or its derivative, colcemid, thereby inhibiting the assembly of tubulin to form mitotic spindles during mitotic process. Therefore it requires cells to pass through the mitosis. However, it is well known by those skilled in the art, that it is often difficult to obtain chromosomes in the case in which cells are not proliferate well.
Furthermore, many phenomena occur during interphase; for example, chromosomal cleavage by irradiation and subsequent reunion occur through the interphase nuclei, resulting in chromosomal aberration. So it has been required to establish the technique that allows one to obtain chromosomes from interphase cells.
The first approach was done by Johnson and Rao (Johnson and Rao, 1970, Nature 226:717). They fused interphase cell in mitotic cell, resulted in the interphase chromosomes condense prematurely (Premature Chromosome Condensation; PCC). However, this method of inducing PCC is technically demanding; synchronizing and collecting large amounts of mitotic cells followed by fusion with target cells. In addition, mixture of chromosomes of interphase and mitotic cell makes it often difficult to analyses the chromosomes of interphase cells.
A similar premature chromosome condensation can be induced by caffeine in cells blocked in S-phase (Schlegel and Pardee, 1986, Science:232, 1264). More recently, okadaic acid has also been reported to induce PCC in cells blocked in S-phase (Ghosh et al., 1992, Exp. Cell Res.:201, 535, Steinmann et al., 1991, Proc. Natl. Acad. Sci. USA:88, 6843, Yamashita et al., 1990, EMBO J:9, 4331). However, the requirement of block the cells in S-phase has limited the use of PCC technique for the analysis of chromosomes.